Studies on the synthesis and anti-Osteoporosis of estrogen-GHRPs linkers.

Article abstract of DOI:10.1016/S0960-894X(02)00847-8

The linkers of estrogen-GHRPs were prepared by the combination of estradiol, estrone, TyrGlyGlyPheLeuOH, and TyrGlyGlyPheLeuOH. Their anti-osteoporosis effect was evaluated by analyzing the data, for instance the weight of the body, femur, femur ash, the content of calcium and phosphor in the femur, the content of calcium and ALP activity in the serum, obtained from the corresponding bioassay in vivo. The results indicated that the anti-osteoporosis potency for estradiol, estrone, TyrGlyGlyPheLeuOH and TyrGlyGlyPheLeuNH(2) may be totally enhanced each other via the corresponding linkers.

Full text of DOI:10.1016/S0960-894X(02)00847-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 143–146
Studies on the Synthesis and Anti-Osteoporosis of
Estrogen-GHRPs Linkers
Chao Wang, Weina Cui, Ming Zhao, Jian Yang and Shiqi Peng*
College of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China
Received 15 August 2002; accepted 11 September 2002
Abstract—The linkers of estrogen-GHRPs were prepared by the combination of estradiol, estrone, TyrGlyGlyPheLeuOH, and
TyrGlyGlyPheLeuOH. Their anti-osteoporosis effect was evaluated by analyzing the data, for instance the weight of the body,
femur, femur ash, the content of calcium and phosphor in the femur, the content of calcium and ALP activity in the serum,
obtained from the corresponding bioassay in vivo. The results indicated that the anti-osteoporosis potency for estradiol, estrone,
TyrGlyGlyPheLeuOH and TyrGlyGlyPheLeuNH2 may be totally enhanced each other via the corresponding linkers.
# 2002 Elsevier Science Ltd. All rights reserved.
It is commonly accepted that the decrease of skeletal
muscle and bone mass depends on the lower level of
estrogen and growth hormone (GH).1,2 Thus supple-
ment of growth hormone releasing peptides (GHRPs)
and estrogen may prevent osteoporosis of elderly
women.3,4 The interaction between estrogen and growth
hormone-insulin like growth factor (GH-IGF) axis
observed so far, for instance estrogen effected obviously
on the secretion of human growth hormone (hGH),
encourage us studying the synthesis and analgesic effects
of kyotorphin-steroid linkers.5 The phenomenon that
the effects of the peptides were enhanced by steroids
indicated that this kind of combination provided an
useful way to find the lead compounds. In the present
paper TyrGlyGlyPheLeuOH, and TyrGlyGlyPhe-
(DCC) and 1-hydroxybenzotriazole (HOBt) were used as
the coupling agents in the peptide formation, hydrogen
chloride in ethyl acetate was used as the deprotective
agent in the removal of Boc, the yields for all of the
reactions were in 77–90%. At 0 ꢀC 1 was treated with 2
mol/L of NaOH and hydrochloric acid successively to
offer the corresponding tetrapeptide acid (2) in 96%
yield. Under the normal conditions 2 was coupled with
l-LeuOMe and l-LeuNH2 to give 3 and 6 in 73 and
76% yield, respectively. Removal of Boc with 4 mol/L
of hydrogen chloride in ethyl acetate 3 and 6 were con-
verted into 4 and 7 in 96% yield. Saponification and
acidification of 3 gave 5 in 88% yield. In the presence of
Pd/C (10%), H2, and 4.4% of formic acid in methanol 4
and 7 were converted into 8 and 9 in 94 and 91% yield,
respectively (Scheme 1).
6
LeuNH2 were coupled with estradiol and estrone to
simulate their enhanced anti-osteoporosis effect via the
corresponding estrogen-GHRPs.
The O-alkylation of estrone with ethyl bromoacetate
and sodium ethoxide provided ethyl estrone-3-oxy-
methylenecarboxylated (10) in 83% yield. Treating 10
with 2 mol/L of NaOH and 2 mol/L of hydrochloric
acid 11 was obtained in 94% yield, which was coupled
with 4 and 7 in the presence of DCC and HOBt to offer
3-protective pentapeptide-estrone (12a,b) in 64 and 85%
yield, respectively. In the presence of Pd/C (10%), H2,
and 4.4% of formic acid in methanol 12a,b were con-
verted into the C-terminal protective 3-pentapeptide-
estrone (13a,b) in 92 and 90%, yield, respectively Sapo-
nification and acidification of 13a gave the goal linker
14a in 51% yield. Reducing 12b and 14a with sodium
Chemistry
The protective tetrapeptide (1) was prepared by use of
stepwise synthesis (from C terminal to N terminal) via
solution method in which dicyclohexylcarbodiimide
*Corresponding author. Tel.: +86-10-6209-2482; fax: +86-10-6209-
2311; e-mail: sqpeng@tree.bjmu.edu.cn
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00847-8

144
C.Wang et al./ Bioorg.Med.Chem.Lett.13 (2003) 143–146
Scheme 1. (I) With DCC, HOBt as the coupling agent; HCl/CH3CO2C2H5 as the deprotection agent of Boc; (II) 2 mol/L of NaOH, and 4 mol/L of
HCl, 0 ꢀC; (III) DCC, HOBt LeuOCH3; (IV) 4 mol/L of hydrochloride in ethyl acetate; (V) 2 mol/L of NaOH, 0 ꢀC; (VI) DCC, HOBt LeuNH2;
(VII) 4 mol/L of hydrochloride in ethyl acetate; (VIII) Pd/C (10%), H2 and 4.4% of HCOOH in CH3OH.
Scheme 2. (I) NaOEt, C2H5OH, BrCH2CO2C2H5; (II) 2 mol/L of NaOH; (III) DCC, HOBt, NMM, 4, 7; (IV) Pd/C (10%), H2 and 4% of HCOOH
in CH3OH; (V); NaBH4; (VI) 2 mol/L of NaOH; in 12a and 13a R=OCH3 in 12b and 13b R=NH2.
borohydride gave 3-protective pentapeptide-estradiol
(15b and 16a) in 92 and 94% yield, respectively. Using
the same reaction conditions as that for 12a,b 1 5bwas
converted into the goal linker 16b in 91% yield (Scheme 2).
Acylation of the 17-hydroxyl in estradiol with succinic
anhydride and DMAP estradiol-17-oxycarbonylpro-
pionic acid (17) was obtained in 66% yield. Treating 17
with 7 and DCC estradiol-17-oxycarbonylpropionyl-
protective pentapeptide amide (18) was provided in
70% yield, which was deprotected in the presence of
Pd/C (10%), H2, and 4.4% of formic acid in methanol
to give estradiol-17-oxycarbonylpropionylpentapeptide
amide (19) in 90% yield (Scheme 3).
In the presence of DCC and DMAP estradiol was acyl-
ated by 5 and the corresponding protective pentapepti-
dyl-17-O-estradiol (20) was obtained in 67% yield.
Treating 20 with Pd/C (10%), H2, and 4.4% of formic
acid in methanol, and 4 mol/L of hydrogen chloride in
Scheme 3. (I) Succinic anhydride, DMAP; (II) DCC, HOBt, NMM
and 7; (III) Pd/C (10%), H2 and 4.4% of HCOOH in CH3OH.

C.Wang et al./ Bioorg.Med.Chem.Lett.13 (2003) 143–146
145
ethyl acetate successively 22 was obtained in 88% yield
(Scheme 4).
normal food (containing 1.76% of calcium and 1.01%
of phosphor) and running water, and injected 2 mL of
NS once a day. All of the animals were treated accord-
ing to the corresponding procedure for 4 weeks after
which their weight of body, femur, and femur ash, the
content of calcium and phosphor in the femur, the level
of calcium and the activity of alkaline phosphatase
(ALP) in the serum were tested. The results are listed in
Tables 1–3. The statistical analysis of the data were
carried out by use of ANOVA test; p<0.05 was
considered significant.
Bioassay in vivo7
Male ICR rats, weighting 20Æ2 g, housed in a 12/12
light/dark cycle at 21Æ2 ꢀC for 24 h before being used.
Except the normal saline (NS) group the animal was
administrated special food (containing 0.1% of calcium
and 0.4% of phosphor) and distilled water, and injected
2 mL of the solution of prednisone (6.3 mg/kg) in NS
intramuscularly twice a week and 2 mL of NS once a day
for the prednisone control group, and injected 2 mL of
the solution of prednisone (6.3 mg/kg) in NS intra-
muscularly twice a week and 2 mL of the solution of
estradiol, or estrone, or GHRPs or estrogen-GHRPs
linkers (18 mg/kg) in NS once a day for the drug receiv-
ing groups. For NS group the animal was administrated
Discussion
In the preparation of TyrGlyGlyPheLeuOH, TyrGly-
GlyPheLeuNH2 and the corresponding estrogen-
GHRPs the Cl2Bzl (2,6-dichlorobenzyl) groupin Tyr
Table 2. Content of calcium and phosphor in femur
GroupCalcium (%) hor (%)
Phosp
NS
Prednisone
Estradiol
Estrone
8
47.85Æ5.28
22.24Æ2.16
20.87Æ2.36
21.85Æ2.43
22.02Æ2.28
22.27Æ2.71
22.35Æ3.50
23.12Æ2.39c
23.48Æ3.04c
24.33Æ.07d,e,g
25.10Æ3.08a,e
25.24Æ2.92a,f,h
23.40Æ2.44c
39.54Æ6.07b
42.11Æ5.75a
40.83Æ4.75b
43.75Æ6.42
43.97Æ6.10
45.30Æ9.79
46.94Æ8.50
47.73Æ6.80
48.94Æ6.15
48.63Æ7.80
46.03Æ6.13
9
14a
13b
16a
16b
19
22
N=12; NS=vehicle; (a) compare to NS, p<0.05; (b) compare to NS,
p<0.01; (c) compare to prednisone, p<0.05; (d) compare to pred-
nisone, p<0.001; (e) compare to estradiol, p<0.05; (f) compare to
estradiol, p<0.01; (g) compare to 8, p<0.05; (h) compare to 9,
p<0.05.
Scheme 4. (I) DCC, HOBt, NMMand 7; (II) Pd/C (10%), H2 and
4.4% of HCOOH in CH3OH; (III) 4 mol/L of hydrochloride in ethyl
Table 1. Weight of the body, femur and femur ash
Table 3. Content of calcium and ALP activity in serum
GroupBody (g)
Femur (mg)
Femur ash (mg)
GroupC alcium (mmol/L)
ALP
NS
Prednisone
Estradiol
Estrone
8
39.40Æ2.27
44.75Æ3.96
27.21Æ2.71
31.89Æ2.68c
35.39Æ3.40a,e
33.62Æ2.90c
33.29Æ2.29c
34.16Æ2.14c,d
34.27Æ2.98c,d
35.83Æ3.16b,e
36.46Æ2.18b,f,n
38.00Æ2.01f,g,q
37.24Æ3.93f,o
36.87Æ3.16f,n
33.39Æ3.01c
21.26Æ1.67c
NS
Prednisone
Estradiol
Estrone
8
1.91Æ0.25
43.50Æ7.91
35.09Æ3.97c
22.82Æ3.84b
2.42Æ0.32c
2.13Æ0.29d
2.28Æ0.30b
2.18Æ0.21a,d
2.01Æ0.20e
2.00Æ0.13f,j,l
1.99Æ0.27f,i
1.95Æ0.24f,l
1.90Æ0.15f,g
1.92Æ0.21f
1.94Æ0.13f,m
48.70Æ7.35
33.93Æ3.81c
21.69Æ1.79c
44.10Æ8.43
39.49Æ3.27b,f,h,k
40.27Æ3.37b,f,h,k
40.60Æ4.41a,f,j
41.68Æ3.04a,f,k
42.64Æ3.62f,m,l
43.68Æ3.22f,m,o
42.56Æ4.31f,m,n
41.13Æ4.23a,f,h
21.74Æ2.89c
45.84Æ8.03
22.80Æ2.73b
23.75Æ2.19b,d,j
24.90Æ2.46a,f,k
25.36Æ1.70f,h,m
26.39Æ3.31f,I,n
25.46Æ2.85f,g,n
24.84Æ2.79a,f,l
37.70Æ9.52e
9
9
34.22Æ8.50a,f,g
32.01Æ7.02b,f,k
30.89Æ9.04b,f,k
29.92Æ8.07c,f,h,j
28.10Æ8.08c,f,h
28.24Æ7.92c,f,h
29.40Æ8.44c,f,h,l
14a
13b
16a
16b
19
14a
13b
16a
16b
19
22
22
N=12; NS=vehicle; (a) compare to NS, p<0.05; (b) compare to NS,
p<0.01; (c) compare to NS, p<0.001; (d) compare to prednisone,
p<0.05; (e) compare to prednisone, p<0.01; (f) compare to pred-
nisone, p<0.001;(g) compare to estradiol, p<0.05; (h) compare to
estradiol, p<0.01; (i) compare to estradiol, p<0.001; (j) compare to
estrone, p<0.05; (k) compare to estrone, p<0.001; (l) compare to 8,
p<0.05; (m) compare to 8, p<0.01; (n) compare to 9, p<0.05; (o)
compare to 9, p<0.01.
N=12; NS=vehicle; (a) compare to NS, p<0.05; (b) compare to NS,
p<0.01; (c) compare to NS, p<0.001; (d) compare to prednisone,
p<0.05; (e) compare to prednisone, p<0.01; (f) compare to pred-
nisone, p<0.001; (g) compare to estradiol, p<0.05; (h) compare to
estradiol, p<0.001; (i) compare to estrone, p<0.05; (j) compare to
estrone, p<0.01; (k) compare to estrone, p<0.001; (l) compare to 8,
p<0.05; (m) compare to 8, p<0.001.

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C.Wang et al./ Bioorg.Med.Chem.Lett.13 (2003) 143–146
residue of all the intermediates, some of which are
unstable to HF, can be removed in satisfactory yield in
the presence of Pd/C (10%), H2, and 4.4% of formic
acid in methanol. The data in Table 1 indicated that the
prednisone induced weight decrease of the body, the
femur and the femur ash of the rats was obviously
improved by 13b, 14a, 16a, 16b, 19 and 22 treating. The
differences of the weights between estradiol, 8, 16a and
22 groups; between estradiol, 9, 16b and 19 groups;
between estrone, 8 and 14a groups; and between estrone,
9 and 13b groups suggested that the anti-osteoporosis
potency of estrogen and GHRPs was enhanced each
other. The data in Table 2 indicated that the content of
calcium in the femur of the rats treated with NS or 8, 9,
13b, 14a, 16a, 16b, 19, 22 gave no significant difference.
The phosphor level in the femur of the rats treated with
13b, 14a, 16a, 16b, 19 and 22 was obviously higher than
the others. The data in Table 3 indicated that the con-
tent of calcium in serum of the rats treated with 13b,
14a, 16a, 16b, 19 and 22 was reduced to the level of NS
treating rats and their ALP activity was significantly
lower than that of normal rats. All of the results
obtained here showed that the combination of estrogen
and GHRPs may be an useful way to find the corre-
sponding lead compounds.
Acknowledgements
The author Shiqi Peng wishes to thank the Key Basic
Research Project (G1998051111) of China for financial
support.
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